With an increased risk of terrorist attacks involving chemical weapons, including nerve agents, there is a need to develop new and improved countermeasures for the rapid detection of exposure. Hence, this project will develop a portable micro-analytical system for the rapid evaluation of exposure to organophosphorus (OP) nerve agents utilizing phosphorylated cholinesterase (ChE) as a biomarker (OP-ChE adducts) in blood and saliva. Our hypothesis is that a highly selective, ultra sensitive, and disposable biosensor can be developed to detect sub-clinical exposure to nerve agents by using nanoparticle linked antibodies and metal chelation to selectively capture OP-ChE biomarkers followed by electrochemical detection. This project is directly responsive to RFA-NS-06-004 concerning CounterACT research projects (U01) for the development of new diagnostic techniques and tools to enhance our medical response capabilities during an emergency. The approach will involve initial development of an immuno-capture electrochemical detector for OP-ChE biomarkers and the subsequent development of a disposable immunochromatographic electrochemical biosensor (IEB). Diisopropylphosphorofluoridate (DFP) and Sarin will be used as the chemical nerve agents in in vitro and in vivo exposures to generate OP-ChE protein biomarkers in the plasma, RBC, and saliva of rats and humans. OP-ChE biomarkers will be detected with the microfluidic IEB using an approach similar to the ELISA assay, except that primary recognition is performed by nanoparticle-tagged antibodies and secondary recognition is performed by zirconium oxide (ZrO2) chelation with the phosphorylated moiety, followed by detection that is based on generating metal ions by dissolving the nanoparticle and detecting the metal ions upon accumulation to an electrode. The development of a real-time biomonitoring IEB device for analysis of blood and/or saliva represents a significant advancement over current biomonitoring strategies for nerve agent exposure. The utilization of minimally invasive biomatrices, coupled with on-site and rapid detection represents a novel approach with broad application for evaluating deliberate or accidental exposures to chemical nerve agents.